Gene delivery has great potential, both as a therapeutic to treat disease on the genetic level and as a technology to facilitate regenerative medicine. One challenge to gene delivery is finding a safe and effective delivery system. Because viral gene therapy can have serious safety concerns, many recent efforts have focused on non-viral gene delivery methods that use biomaterials. Many biomaterials, including cationic lipids, sugars, peptides, and polymers, have been shown to be effective for delivering genes in vitro. See T. G. Park, et al., Adv. Drug Del. Rev. 58:467-486 (2006); D. W. Pack, et al., Nat. Rev. Drug Discovery 4:581-593 (2005); and M. C. Pedroso de Lima, et al., Adv. Drug Del. Rev. 47:277-94 (2001).
One of the lead polymers for gene delivery is polyethylenimine (PEI), which, due its cationic structure, can be very effective for binding DNA and forming gene delivery particles. See O. Boussif, et al., Proc. Natl. Acad. Sci. USA 92:7297-301 (1995). PEI also is particularly effective at promoting endosomal escape of PEI/DNA particles through the proton sponge mechanism. See N. D. Sonawane, et al., J. Biol. Chem. 278:44826-44831 (2003); A. Akinc, et al., J. Gene Med. 7:657-663 (2005). This mechanism is critical in preventing lysosomal degradation of the DNA and to enable efficient delivery of the DNA to the cytoplasm. This endosomal escape mechanism has been used in the design of other synthetic gene delivery polymers, including polylysine-based polymers that contain an imidazole group in the side chain. See D. Putnam, et al., Proc. Natl. Acad. Sci. USA 98:1200-5 (2001). Although PEI shows promise compared to other biomaterials, it also leads to significant cytotoxicity, see S. M. Moghimi, et al., Mol. Ther. 11:990-5 (2005), and has lower effectiveness than viral methods.
One newer group of polymers used for gene delivery are poly(beta-amino ester)s, see J. J. Green, et al., Acc. Chem. Res. 41:749-759 (2007), which are useful due to their ability to bind DNA, promote cellular uptake, facilitate escape from the endosome, and allow for DNA release in the cytoplasm. See D. M. Lynn and R. Langer, J. Am. Chem. Soc. 122:10761-10768 (2000); D. G. Anderson, et al., Mol. Ther. 11:426-34 (2005); and A. Akinc, et al., J. Am. Chem. Soc. 125:5316-23 (2003). Unlike PEI, poly(beta-amino ester)s are readily biodegradable due to their ester linkages, which reduces cytotoxicity. D. M. Lynn and R. Langer, supra, J. J. Green, et al., Bioconjugate Chem. 17:1162-1169 (2006). It has been shown that within this class, acrylate-terminated polymers have low gene delivery, whereas amine monomer-terminated polymers have higher delivery. See A. Akinc, et al., Bioconjugate Chem. 14:979-988 (2003). Recently, end-modification with diamine monomers has shown that some of these polymers can rival adenovirus for gene delivery in vitro and also are effective in vivo. See J. J. Green, et al., Adv. Mater. 19:2836-2842 (2007); G. T. Zugates, et al., Mol. Ther. 15:1306-1312 (2007).
Another approach to increase gene delivery effectiveness while reducing cytotoxicity involves adding bioreducible linkages to polymers. Disulfide linkages have been added to PEI to produce bioreducible versions with lower cytotoxicity than high molecular weight versions of the parent polymer. M. A Gosselin, et al., Bioconjugate Chem. 12:989-994 (2001); M. L. Forrest, et al., Bioconjug Chem. 14:934-40 (2003). Other researchers have shown that bioreducible poly(amido amines) can have higher efficacy than PEI while also having reduced cytotoxicity. See L. V. Christensen, et al., Bioconjugate Chem. 17:1233-40 (2006); C. Lin, et al., J. Controlled Release 126:166-74 (2008).
Further, it was recently demonstrated that IMR-90 human primary fibroblasts can be reprogrammed to induced pluripotent stem cells with integrating viruses. See J. Yu, et al., Science 318:1917-20 (2007). Reprogramming human differentiated cells into undifferentiated, pluripotent cells could potentially allow a patient to receive a customized cell therapy that is a perfect genetic match.